Leucine-rich repeats (“LRRs”) were first discovered in leucine-rich α2-glycoprotein isolated from human serum (Takashashi, et al. (1985) Proc. Natl. Acad. Sci. USA 82:1906–1910). LRR-containing proteins represent a diverse group of molecules with differing functions and cellular locations in a variety of organisms (for review, see Kobe and Deisenhofer (1994) Trends Biochem. Sci. 19:415–421).
Given the wide range of important functions of LRR-containing proteins, such as protein:protein interactions, matrix association, cell adhesion, caspase recruitment (CARD), nucleotide binding, and signal transduction, there exists a need for identifying novel LRR containing proteins as well as for modulators of such molecules for use in regulating a variety of cellular responses. Proteins within this group have also been found to play a role in cell adhesion during various developmental processes. Adhesion proteins represent the largest group in the LRR superfamily.
As the name implies, LRRs are distinguished by a consensus sequence containing predominently of leucines. LRRs are short protein modules characterized by a periodic distribution of hydrophobic amino acids, especially leucine residues, separated by hydrophilic residues (Sean et al., Prog. Biophys. Molec. Biol. 65:1–44, 1996). The basic structure of the repeat is as follows: X-L-X-X-L-X-L-X-X-N-X-a-X-X-X-a-X-X-L-X (SEQ ID NO:75), where X is any amino acid, L is leucine, N is asparagine and “a” denotes an aliphatic residue, such as glycine, alanine, valine, leucine and isoleucine. The asparagine at position 10 can be replaced by cysteine, threonine, or glutamine. The average repeat length is 24 amino acids but can vary between 22 and 29 amino acids. In transmembrane proteins, LRRs and their flanking sequences always occur in the presumed extracellular portions. In these situations, the LRRs are generally flanked on either side by cysteine-rich regions. Generally, the cysteines are present in the oxidized disulphide link form.
A class of cell surface proteins, having a leucine-rich repeat (LRR) in the carboxy terminus of the polypeptide chain, has been described in both plants and animals that are involved in pathogen perception, MHC class II trans-activation, inflammation, and the regulation of apoptosis (Dixon et al., Proc. Natl. Acad. Sci, USA. 97:8807–14:2000; Harton and Ting, Mol. Cell. Biol. 20:6185–6194:2000; Inohara et al., J. Biol. Chem. 275:27823–27831, 2000; Inohara et al., J. Biol. Chem. 274:14560–14567, 1999).
An example of a transmembrane protein containing an LRR is Toll, a Drosophila gene that functions to establish dorsal-ventral patterning. Dominant ventralizing mutants that map to the cysteine-rich regions surrounding the LRR domain have been described (Schneider et al., Genes and Development 5:797–807, 1991). The cysteine regions associated with LRRs act to regulate receptor activity. The LRRs, within the Toll protein, have been shown to function in heterotypic cell adhesion, a process required for proper motoneuron and muscle development (Halfon et al., Dev. Biol. 169:151–167, 1995).
Another Drosophila LRR-containing transmembrane protein, 18 wheeler, which is regulated by homeotic genes, also promotes heterophilic cell adhesion in cell migration events during development (Eldon et al., Development 120:885–899, 1994). Mammalian CD14, which binds lipopolysaccharide (LPS), and signals through NF-κB, is thought to have analogies to the Toll signal transduction pathway. CD14 also contains a region of LRRs that has been shown in deletion mutants to be responsible for LPS binding.
Slit is another LRR-containing Drosophila secreted protein that functions in the development of the midline glial cells and the commissural axon tracts that cross the midline (Jacobs and Goodman, J. Neurosci. 9:2402–2411, 1989). Slit is secreted by midline glia and forms a gradient by diffusion. Another protein, Robo, responds to the Slit gradient and specifies the lateral position of axons in developing the central nervous system (Simpson et al., Cell 103:1019–1032, 2000). Mammalian homologues of Drosophila Slit have been shown to bind the heparin sulfate proteoglycan, glypican-1 (Liang et al., J. Biol. Chem. 274:17885–1792, 1999). In general, heparin sulfate proteoglycans have been shown to accummulate in Alzheimer's diseased brains and specifically, glypican-1 is a component of both senile plaques and neurofibrillary tangles (Verbeek et al., Am. J. Pathol. 155:2115–2125, 1999). Heparin sulfate proteoglycans are also implicated in the regulation of cytokine signaling in B cells through the activation of CD40 (van Der Voort et al., J. Exptl. Med. 192:1115–1124, 2000).
Direct evidence that mutations in proteins that contain LRRs can lead to human disease has recently been demonstrated. For example, one of the genes associated with susceptibility to Crohn's disease, NOD2, a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators, contains mutations that alter the structure of either the LRR or the adjacent region (Hugot et al., Nature 411:599–603, 2000). Other examples are mutations in a pyrin-like LRR-containing gene that causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome (Hoffman et al., Nature Genetics 29:301–305, 2001).
LRR-containing proteins have been identified in prokaryotes, plants, yeast, and mammals. Although these proteins were initially thought to be secreted proteins, it is now appreciated that they inhabit a variety of cellular locations and participate in a diverse set of critical functions in developmental and cellular homeostasis. These LRRs, being extracellular, are capable of directing protein-protein interactions with other receptors that are involved in regulating developmental processes, apoptosis, inflammation, and immune responses. LRR-containing proteins can also bind to other extracellular ligands derived from infectious agents and participate in triggering and or modulating immune responses. Therefore, agonists and antagonists for these LRR-containing proteins can be useful for therapeutic purposes.